It is of importance to be able to sequence certain molecules, e.g. sequences of nucleic acids, proteins (poly-) and other complex biomolecular entities, for example in order to be able to do diagnostic screening.
In literature it is proposed that solid-state nanopores can be used for biochemical analysis {Dekker2007}, mostly structural analysis of linear organic molecules. A particular application that is often quoted is DNA sequencing {Deamer2000}. Solid-state nanopores are holes fabricated artificially in a membrane with diameter in the range (0.1 nm-999 nm). Molecular sequencing in such nanopore relies on translocation of the target molecule through the nanopore and subsequent transduction via highly localized physical interactions with the section of the molecule in the pore leading to measurable signals.
Transduction and recognition are performed sequentially and in real-time on segments of the molecule. Translocation is achieved passively or (with greater control) actively. Active translocation can be achieved by means of electrophoresis in which a voltage is applied on (two) electrodes placed in fluidic reservoirs separated by the membrane, the resulting electrical field then propels the charged molecule through the pore. In other examples optical or magnetic forces are exerted on the molecule or on bead(s) attached to the molecule to stimulate translocation or to modulate the translocation speed.
Various electric or electronic interactions can be exploited for sensing in the pore. For chemical analysis, chemically specific interactions are investigated. DNA translocation events are routinely detected by measurement of the ion current through the nanopore. The presence of a DNA molecule in the pore leads to an increase or decrease of the ionic current. Provided such measurements can be performed with sufficient sensitivity, information on structural or chemical composition of the molecule could be harvested from ionic current data. In another method, electrodes are mounted in the pore and electronic properties of the molecule are measured there. When a voltage is applied across the electrodes, an electronic current can flow stimulated by quantum mechanical electron tunneling via the electronic states of the molecule. Such mechanism provides chemical specificity. In yet another approach, capacitive modulations are sensed. {Dekker2007}specifically discloses a method called force spectroscopy on DNA in nanopores. Here, one end of the DNA is attached to a bead, which is trapped in the focus of an infrared laser. Subsequently, individual DNA molecules are inserted into a single nanopore and the DNA is arrested during voltage-driven translocation. The force acting on the DNA then pulls the bead away from the centre of the optical trap. This can be measured with high accuracy using the reflected light from the bead.
However all these methods are in need of improvement in order to obtain a more reliable result. Particularly, the force spectroscopy requires coupling between one end of a DNA molecule and a bead. Such coupling is an additional step that is undesired when intending to apply the method on a large scale for diagnostic use. Moreover, Dekker effectively senses that a bead is pulled away from the centre. There may be different causes, including side effects, impact of contaminants (e.g. other molecules present), viscosity and mass and heat transfer effects. Such different causes likely tend to reduce robustness and/or signal to noise ratio of the measurement.
WO03/16781 discloses a method of analyzing molecules such as DNA, wherein light is directed to a metal surface of a membrane having one or more apertures. Sidewalls of the apertures may be covered with metal, or alternatively, a thin, annular metallic ring may be present on the opposing surface of the substrate. The incident light excites surface plasmons (electron density fluctuations) in the top metal surface and this energy couples through the apertures to the opposing surface where it is emitted as light from the apertures or from the rims of the apertures. The extent to which surface plasmons are induced on the surface at the aperture exit may be limited, thereby constraining the resulting emissions to small target areas. The resulting spot illumination may be used to analyze the properties of small objects such as proteins and nucleic acid molecules and single cells.
The WO03/16781 discloses in its figure description more precisely how such analysis may be carried out. In one embodiment disclosed on page 39 and beyond, the document specifies that surface plasmon enhanced illumination can be used for implementation of an array based technique to study macromolecules and their interactions in solution, and to investigate cell surface phenomena in intact cells. The array can study different unlabeled macromolecules in parallel. The technique identifies the molecule using signatures that are isolated within a rich data set that is based on the macromolecules' interactions that yield measurable photonics effects or signatures. Detecting signatures is based on detecting changes in the emission spectra from the apertures indicating the presence and identity of the molecules affecting the changes. Light intensity may be detected with a CCD-detector. Alternatively, the device may be illuminated with monochromatic light that is scanned across the UV-visible-IR portion of the electromagnetic spectrum and the intensity monitored as the wavelength is scanned. Alternatively, shifts in resonance frequency may be detected.
It is observed that data shown in FIG. 29-32 of WO03/016781 do not reflect actual data. They are merely illustrative of the manner in which molecular characteristics may be manifested by intensity data, as acknowledged on page 42.
Furthermore, the WO03/016781 mentions the option of having ligands bound to the illumination side of the apertures and thereafter measure changes in intensity or wavelength. Here, it appears that the intended measurement is particularly a measurement of the number of devices. As acknowledged on page 50, first paragraph, the arrangement will display behaviour similar to a Geiger counter showing a count and a rate.
It is a disadvantage of WO03/016781 that it is after all not clear, how to obtain signatures that could be linked to the molecular structure. Moreover, the idea that identification of signatures could be based on comparison of a measured signal with a rich data set appears quite theoretical. The amount of macromolecular interactions yielding measurable photonics effects appears to be huge, and further appears in need for correction due to environmental effects. The analogy with the Geiger teller additionally suggests that the method of WO03/016781 is more suitable for counting molecules rather than for identification of the structure of the individual molecules.